Carbonic anhydrases (CA) are a family of 16 distinct but related metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) and protons (H+) (Pastorekova et al., 2004; see FIG. 1). Members of the CA, with the exception of CA-IX and CA-XII, can be found in many normal human organs, tissues and subcellular compartments where they play an important role in the regulation of the extracellular and intracellular pH (pHe and pHi, respectively) and the secretion of electrolytes (Zatovicova et al., 2005; Thiry et al., 2006).
In addition to its pH-balancing activities, CA-IX has been shown to be involved in cell adhesion and migration (Svastova et al., 2011) and has been associated with cancer progression, metastasis and poor clinical outcome (Neri et al., 2011). CA-IX (also known as MN, P54/58N or Renal Cell Carcinoma (RCC)-associated protein G250) is a transmembrane protein with an extracellular catalytic site and an NH2-terminal proteoglycan (PG)-like domain. The C-terminal intracellular portion of CA-IX is involved in the inside-out regulation of the extracellular catalytic domain through the phosphorylation of Thr-443 by protein kinase A (PKA) (Hulikova et al., 2009; Ditte et al., 2011). Expression of CA-IX is tightly controlled by hypoxia-inducible factor 1 alpha (HIF-la). CA-IX is expressed on the surface of tumor cells located in pre-necrotic areas of tumors (Wykoff et al., 2000) where it is involved in promoting tumor cell survival, the accelerated degradation of the extracellular matrix (ECM) and metastasis.
CA-IX has a very selective expression pattern in normal tissue. The mucosa of the gall bladder and stomach express high levels of CA-IX. Low expression levels of CA-IX levels can be found in the intestinal epithelium, and even lower levels in pancreatic duct epithelium, male reproductive organs, and cells that line the body cavity. All other normal tissues do not express CA-IX. Cancerous tissues however, especially those of the cervix, kidney and lung, express high levels of CA-IX thus making CA-IX a very attractive therapeutic tumor target. While various small molecule inhibitors have been shown to effectively inhibit the catalytic activity of CA-IX (Supuran et al., 2008; Neri et al., 2011; Pacchiano et al., 2010; Lou et al., 2011), the lack of target specificity has been an ongoing challenge.
In order to address this issue and to confer specificity in targeting CA-IX, various antibodies have been raised against this important target.
One of the earliest monoclonal antibodies (mAb) raised against CA-IX is M75 (Pastorekova et al., 1992), which binds to CA-IX's PG-like domain. M75 has been predominately used as tool for CA-IX detection in vitro and in vivo (Chrastina et al., 2003a, 2003b; Zatovicova et al., 2010).
A second anti-CA-IX mAb, mAb G250 (Oosterwijk et al., 1986), was shown to interact with CA-IX's catalytic domain without however inhibiting its enzyme activity. A chimeric version of G250 (designated cG250) was developed as a therapeutic antibody (Surfus et al., 1996; Oosterwijk, 2008) with a mechanism of action that was shown to rely predominantly on an Antibody-Dependent Cellular Cytotoxicity (ADCC) response. cG250 does however not improve the disease-free survival rate of patients (>6-year span) compared to a placebo (Bleumer et al., 2004). Despite the lack of therapeutic potential of the cG250 antibody itself, the mAb continues to be developed for the treatment of cancer in combination with IL2 or IFN-α, as an imaging diagnostic agent and for in vitro diagnostics (IVD) immunohistochemistry (IHC) assays.
In addition, cG250 is also used as a vehicle for the delivery of radionuclides. Specifically, Brouwers et al. (2004) successfully used cG250 to shuttle 177Lu and 90Y into tumor cells, causing growth retardation of xenograft tumors. Clinical phase II/III studies with these labeled mAbs are currently underway (Stillebroer et al., 2012). Also in development are antibody-drug conjugates (ADC) based on cG250, however little is known their efficacy. Such antibody-drug conjugates are an attractive option in cancer therapy, as they combine the selective targeting ability of the antibody with the cell-killing capabilities of the cytotoxic drug.
In view of its specific tumor expression, CA-IX as a therapeutic target has become an active area of research. Although several antibodies have been identified showing enzyme inhibition, only one has been evaluated in vivo (VII/20 mAb; Zatovicova et al., 2010). Similarly, the use of these mAb for the delivery of cytotoxic agents or radionuclides to tumor cells expressing CA-IX has been an area of much investigative research. For example, Petrul et al. (2012) isolated the 3ee9 Fab, which was subsequently engineered into a mAb and further developed as an ADC by conjugation to monomethyl auristatin E. This ADC showed potent antitumor efficacy and a Phase I clinical trial to determine the maximal tolerated dose (MTD) was terminated early due to safety concerns.
While there is interest and research activity surrounding the use of CA-IX as a target for ADC, there is currently little certainty surrounding ongoing investigations involving these known antibodies. The ability of an antibody to function as an ADC is difficult to predict, and relies on design strategies, target biology and routing behaviour that go beyond its ability to be internalized by its specific target. Therefore, there remains a need in the art to develop further anti-CA-IX antibodies that have potential as ADC candidates. Needless to say such antibodies should display a high target affinity and specific while avoiding off-target effects, toxicity, and therapeutic resistance.